1. Field of the Invention
The present invention relates to a vaccine that treats cancer as well as a vaccine that treats or prevents human immunodeficiency virus (HIV) infection. In particular, the present invention provides a fusion polypeptide comprising a chemokine and either a tumor or viral antigen which is administered as either a protein or nucleic acid vaccine to elicit an immune response effective in treating cancer or effective in treating or preventing HIV infection.
2. Background Art
Tumor cells are known to express tumor-specific antigens on the cell surface. These antigens are believed to be poorly immunogenic, largely because they represent gene products of oncogenes or other cellular genes which are normally present in the host and are therefore not clearly recognized as nonself. Although numerous investigators have tried to target immune responses against epitopes from various tumor specific antigens, none have been successful in eliciting adequate tumor immunity in vivo (71).
Humans are particularly vulnerable to cancer as a result of an ineffective immunogenic response (72). In fact, the poor immunogenicity of relevant cancer antigens has proven to be the single greatest obstacle to successful immunotherapy with tumor vaccines (73). Over the past 30 years, literally thousands of patients have been administered tumor cell antigens as vaccine preparations, but the results of these trials have demonstrated that tumor cell immunization has failed to provide a rational basis for the design or construction of effective vaccines. Even where patients express tumor-specific antibodies or cytotoxic T-cells, this immune response does not correlate with a suppression of the associated disease. This failure of the immune system to protect the host may be due to expression of tumor antigens that are poorly immunogenic or to heterologous expression of specific antigens by various tumor cells. The appropriate presentation of tumor antigens in order to elicit an immune response effective in inhibiting tumor growth remains a central issue in the development of an effective cancer vaccine.
Chemokines are a group of usually small secreted proteins (7-15 kDa) induced by inflammatory stimuli and are involved in orchestrating the selective migration, diapedesis and activation of blood-born leukocytes that mediate the inflammatory response (23,26). Chemokines mediate their function through interaction with specific cell surface receptor proteins (23). At least four chemokine subfamilies have been identified as defined by a cysteine signature motif, termed CC, CXC, C and CX3C, where C is a cysteine and X is any amino acid residue. Structural studies have revealed that at least both CXC and CC chemokines share very similar tertiary structure (monomer), but different quaternary structure (dimer) (120-124). For the most part, conformational differences are localized to sections of loop or the N-terminus.
Monocyte chemotactic protein-3 (MCP-3) is a potent chemoattractant of monocytes and dendritic cells, T lymphocytes, basophils and eosinophils (10, 23, 26, 37).
There remains a great need for a method of presenting tumor antigens, which are known to be poorly immunogenic, xe2x80x9cselfxe2x80x9d antigens to a subject""s immune system in a manner that elicits an immune response powerful enough to inhibit the growth of tumor cells in the subject. This invention overcomes the previous limitations and shortcomings in the art by providing a fusion protein comprising a chemokine and a tumor antigen which can produce an in vivo immune response, resulting in the inhibition of tumor cells. This invention also overcomes previous shortcomings in the field of HIV vaccine development by providing a fusion protein comprising a chemokine and an HIV antigen which is effective as a vaccine for treating or preventing HIV infection.
The present invention provides a fusion polypeptide comprising human monocyte chemotactic protein-3 and human Muc-1, a fusion polypeptide comprising human interferon-induced protein 10 and human Muc-1, a fusion polypeptide comprising human macrophage-derived chemokine and human Muc-1 and a fusion polypeptide comprising human SDF-1 and human Muc-1.
The present invention also provides a fusion polypeptide comprising a human chemokine and a human immunodeficiency virus (HIV) antigen, wherein the chemokine can be IP-10, MCP-1, MCP-2, MCP-3, MCP-4, MIP 1, RANTES, SDF-1, MIG and/or MDC and wherein the HIV antigen can be gp120, gp160, gp41, an active fragment of gp120, an active fragment of gp160 and/or an active fragment of gp41.
In addition, the present invention provides a method of producing an immune response in a subject, comprising administering to the subject any of the fusion polypeptides of this invention, comprising a chemokine and a human immunodeficiency virus (HIV) antigen, or a chemokine and a tumor antigen, either as a protein or a nucleic acid encoding the fusion polypeptide.
Also provided is a method of treating a cancer in a subject comprising adminstering to the subject any of the fusion polypeptides of this invention, comprising a chemokine and a tumor antigen, either as a protein or a nucleic acid encoding the fusion polypeptide.
Further provided is a method of treating or preventing HIV infection in a subject, comprising administering to the subject any of the fusion polypeptides of this invention, comprising a chemokine and a human immunodeficiency virus (HIV) antigen, either as a protein or a nucleic acid encoding the fusion polypeptide.
A method of treating a B cell tumor in a subject is also provided, comprising administering to the subject a fusion polypeptide comprising a human chemokine and a B cell tumor antigen.
Various other objectives and advantages of the present invention will become apparent from the following detailed description.
As used in the claims, xe2x80x9caxe2x80x9d can include multiples. For example, xe2x80x9ca cellxe2x80x9d can mean a single cell or more than one cell.
The present invention is based on the unexpected discovery that the administration of a fusion protein comprising a chemokine and a tumor antigen or administration of a nucleic acid encoding a fusion protein comprising a chemokine and a tumor antigen yields an effective and specific anti-tumor immune response by converting a xe2x80x9cselfxe2x80x9d tumor antigen into a potent immunogen by binding to a chemokine moiety. A further unexpected discovery of the present invention is that the chemokine-scFv fusion polypeptide of this invention is superior to the prototype Id-KLH vaccine in tumor protection studies as described herein.
Thus, the present invention provides a fusion polypeptide comprising a chemokine and a tumor antigen. The fusion polypeptide can be present in a purified form and can induce an immune response against the tumor antigen and inhibit the growth of tumor cells expressing the tumor antigen. xe2x80x9cPurifiedxe2x80x9d as used herein means the polypeptide is sufficiently free of contaminants or cell components with which proteins normally occur to allow the peptide to be used therapeutically. It is not contemplated that xe2x80x9cpurifiedxe2x80x9d necessitates having a preparation that is technically totally pure (homogeneous), but purified as used herein means the fusion polypeptide is sufficiently pure to provide the polypeptide in a state where it can be used therapeutically. As used herein, xe2x80x9cfusion polypeptidexe2x80x9d means a polypeptide made up of two or more amino acid sequences representing peptides or polypeptides from different sources. Also as used herein, xe2x80x9cepitopexe2x80x9d refers to a specific amino acid sequence of limited length which, when present in the proper conformation, provides a reactive site for an antibody or T cell receptor. The identification of epitopes on antigens can be carried out by immunology protocols that are standard in the art (74). As further used herein, xe2x80x9ctumor antigenxe2x80x9d describes a polypeptide expressed on the cell surface of specific tumor cells and which can serve to identify the type of tumor. An epitope of the tumor antigen can be any site on the antigen that is reactive with an antibody or T cell receptor.
As used herein, xe2x80x9cchemokinexe2x80x9d means a small secreted protein, induced by inflammatory stimuli (e.g., fibroblasts, endothelial cells, epithelial cells, monocytes, macrophages, T cells, B cells, PMNs, etc. stimulated by proinflammatory cytokines such as interferon-gamma, interleukin 4, products of Th1 and Th2 lymphocytes, interleukin-1, tumor necrosis factor-alpha and bacterial products such as lipopolysaccharide, as well as viral infection (75,76), which orchestrates a chemotactic response typically after binding to specific G-protein-coupled cell surface receptors on target cells (e.g., antigen presenting cells (APC), such as dendritic cells, monocytes, macrophages, keratinocytes and B cells), comprising the selective migration, diapedesis and activation of leukocytes which mediate the inflammatory response. Four human CXC chemokine receptors (CXCR1-CXCR4), eight human CC chemokine receptors (CCR1-CCR8) and one CXXXC chemokine receptor (CX3CR1) have been identified. As one example, the chemokine, interferon-induced protein 10 (IP-10) binds to the CXCR3 receptor, thus inducing chemotaxis of activated T cells, NK cells, etc., which express this receptor. As another example, the chemokine monocyte chemotactic protein-3 (MCP-3) acts via binding to the CCR1, CCR2 and CCR3 chemokine receptors on antigen presenting cells (APC) such as dendritic cells, eosinophils, basophils, monocytes and activated T cells. Thus, MCP-3 selectively targets and induces chemotaxis of these cell types.
The chemokine of this invention can include, but is not limited to, interferon-induced protein 10, monocyte chemotactic protein-3, monocyte chemotactic protein-2, monocyte chemotactic protein-1, monocyte chemotactic protein-4, macrophage inflammatory protein 1, RANTES, SDF-1, MIG and macrophage-derived chemokine, as well as any other chemokine now known or later identified.
It will be appreciated by one of skill in the art that chemokines can include active fragments of chemokines which retain the chemotactic activity of the intact molecule. For example, for both CC and CXC chemokines, the N terminal region is the critical region of the molecule for biological activity and leukocyte selectivity. In particular, the N-terminal ELR motif-containing CXC chemokines are chemotactic for neutrophils, whereas those not containing the motif act on lymphocytes. IP-10 and MIG, for example, do not contain the ELR motif and are known to attract activated T cells (77). Addition of a single amino acid residue to the amino terminus of MCP-1 decreases its biological activity up to 1000 fold and deletion of a single amino acid for that region converts the chemokine from an activator of basophils to an eosinophil chemoattractant (78).
A chemokine consists of two structural portions: the amino terminal portion and the carboxy terminal portion. The amino terminal portion is responsible for chemokine receptor binding and the carboxy terminal end binds to heparin and heparan sulfate, for example, in the extracellular matrix and on the surface of endothelial cells. The chemokine gene can be fragmented as desired and the fragments can be fused to a specific marker gene encoding an antigen (e.g., Muc-1 VNT, lymphoma scFv, etc.). The fusion polypeptide comprising the chemokine fragment and the tumor or viral antigen can be produced and purified as described herein and tested for immunogenicity according to the methods provided herein. By producing several fusion polypeptides having chemokine fragments of varying size, the minimal size chemokine fragment which impart an immunological effect can be identified.
The tumor antigen moiety of the fusion polypeptide of this invention can be any tumor antigen now known or later identified as a tumor antigen. The appropriate tumor antigen used in the fusion polypeptide naturally depends on the tumor type being treated. For example, the tumor antigen can be, but is not limited to human epithelial cell mucin (Muc-1; a 20 amino acid core repeat for Muc-1 glycoprotein, present on breast cancer cells and pancreatic cancer cells), the Ha-ras oncogene product, p53, carcino-embryonic antigen (CEA), the raf oncogene product, GD2, GD3, GM2, TF, sTn, MAGE-1, MAGE-3, tyrosinase, gp75, Melan-A/Mart-1, gp100, HER2/neu, EBV-LMP 1 and 2, HPV-F4, 6, 7, prostatic serum antigen (PSA), alpha-fetoprotein (AFP), CO17-1A, GA733, gp72, p53, the ras oncogene product, HPV E7 and melanoma gangliosides, as well as any other tumor antigens now known or identified in the future. Tumor antigens can be obtained following known procedures or are commercially available (79). The effectiveness of the fusion protein in eliciting an immune response against a particular tumor antigen can be determined according to methods standard in the art for determining the efficacy of vaccines and according to the methods set forth in the Examples.
Additionally, the tumor antigen of the present invention can be an antibody which can be produced by a B cell tumor (e.g., B cell lymphoma; B cell leukemia; myeloma) or the tumor antigen can be a fragment of such an antibody, which contains an epitope of the idiotype of the antibody. The epitope fragment can comprise as few as nine amino acids. For example, the tumor antigen of this invention can be a malignant B cell antigen receptor, a malignant B cell immunoglobulin idiotype, a variable region of an immunoglobulin, a hypervariable region or complementarity determining region (CDR) of a variable region of an immunoglobulin, a malignant T cell receptor (TCR), a variable region of a TCR and/or a hypervariable region of a TCR.
In a preferred embodiment, the tumor antigen of this invention can be a single chain antibody (scFv), comprising linked VH, and VL domains and which retains the conformation and specific binding activity of the native idiotype of the antibody (27). Such single chain antibodies are well known in the art and can be produced by standard methods and as described in the Examples herein.
In addition, the tumor antigen of the present invention can be an epitope of the idiotype of a T cell receptor, which can be produced by a T cell tumor (e.g., T cell lymphoma; T cell leukemia; myeloma). The epitope can comprise as few as nine amino acids.
As will be appreciated by those skilled in the art, the invention also includes peptides and polypeptides having slight variations in amino acid sequences or other properties. Such variations may arise naturally as allelic variations (e.g., due to genetic polymorphism) or may be produced by human intervention (e.g., by mutagenesis of cloned DNA sequences), such as induced point, deletion, insertion and substitution mutants. Minor changes in amino acid sequence are generally preferred, such as conservative amino acid replacements, small internal deletions or insertions, and additions or deletions at the ends of the molecules. Substitutions may be designed based on, for example, the model of Dayhoff et al. (80). These modifications can result in changes in the amino acid sequence, provide silent mutations, modify a restriction site, or provide other specific mutations. The fusion polypeptides can comprise one or more selected epitopes on the same tumor antigen, one or more selected epitopes on different tumor antigens, as well as repeats of the same epitope, either in tandem or interspersed along the amino acid sequence of the fusion polypeptide. The tumor antigen can be positioned in the fusion polypeptide at the carboxy terminus of the chemokine, the amino terminus of chemokine and/or at one or more internal sites within the chemokine amino acid sequence.
The present invention further provides a polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO:13 (human IP-10 fused to murine scFv38), SEQ ID NO:16 (human MCP-3 fused to murine scFv38), SEQ ID NO:12 (human IP-10 fused to murine scFv20A), SEQ ID NO:14 (human MCP-3 fused to murine scFv20A) SEQ ID NO:1 (human IP-10 fused to human Muc-1 core epitope (VNT)), SEQ ID NO:2 (human MCP-3 fused to human Muc-1 core epitope (VNT)), SEQ ID NO:3 (murine IP-10 fused to human Muc-1 core epitope (VNT)), SEQ ID NO:4 (murine MCP-3 fused to Muc-1 core epitope (VNT)), SEQ ID NO:5 (human SDF-1xcex2 fused to the hypervariable region of the envelope glycoprotein, gp120, of HIV-1 (the disulfate loop V3)), SEQ ID NO:6 (human IP-10 fused to the hypervariable region of the envelope glycoprotein gp120 of HIV-1 (the disulfate loop V3), SEQ ID NO:7 (human MCP-3 fused to the hypervariable region of the envelope glycoprotein gp120 of HIV-1 (the disulfate loop V3), SEQ ID NO:8 (murine IP-10 fused to the hypervariable region of the envelope glycoprotein gp120 of HIV-1 (the disulfate loop V3), SEQ ID NO:52 (human IP-10 fused with HIV gp120), SEQ ID NO:56 (human MCP-3 fused with HIV gp120), and SEQ ID NO:9 (murine MCP-3 fused to the hypervariable region of the envelope glycoprotein gp120 of HIV-1 (the disulfate loop V3). It would be routine for an artisan to produce a fusion protein comprising any human chemokine region and any human tumor antigen (e.g., human single chain antibody) region according to the methods described herein, on the basis of the availability in the art of the nucleic acid and/or amino acid sequence of the human chemokine of interest and the human tumor antigen of interest.
The present invention further provides a fusion polypeptide comprising a first region comprising a chemokine selected from the group consisting of interferon-induced protein 10, monocyte chemotactic protein-2, monocyte chemotactic protein-1, macrophage inflammatory protein 1, RANTES, SDF-1 and macrophage-derived chemokine and a second region comprising a tumor antigen selected from the group consisting of human epithelial cell mucin (Muc-1), the Ha-ras oncogene product, p53, carcino-embryonic antigen (CEA), the raf oncogene product, GD2, GD3, GM2, TF, sTn, MAGE-1, MAGE-3, tyrosinase, gp75, Melan-A/Mart-1, gp100, HER2/neu, EBV-LMP 1 and 2, HPV-F4, 6, 7, prostatic serum antigen (PSA), alpha-fetoprotein (AFP), CO17-1A, GA733, gp72, p53, the ras oncogene product, HPV E7, melanoma gangliosides, an antibody produced by a B cell tumor (e.g., B cell lymphoma; B cell leukemia; myeloma), a fragment of such an antibody, which contains an epitope of the idiotype of the antibody, a malignant B cell antigen receptor, a malignant B cell immunoglobulin idiotype, a variable region of an immunoglobulin, a hypervariable region or CDR of a variable region of an immunoglobulin, a malignant T cell receptor (TCR), a variable region of a TCR and/or a hypervariable region of a TCR.
For example, the present invention provides a fusion polypeptide comprising an scFv cloned from a human subject""s biopsy tumor material or from a hybridoma cell line producing a lymphoma antibody and a human chemokine moiety (e.g., MCP-3, IP-10, SDF-1, etc.). In addition, the present invention provides a human chemokine fused with the Muc-1 core epitope of human breast cancer or human pancreatic cancer. Muc-1 is a glycoprotein (Mr greater than 200,000) abundantly expressed on breast cancer cells and pancreatic tumor cells. A variable number of tandem (VNT) repeats of a 20 amino acid peptide (PDTRPAPGSTAPPAHGVTSA; SEQ ID NO:40) include B and T cell epitopes. Thus, the present invention provides a fusion protein comprising IP-10 and Muc-1 VNT and MCP-3 and Muc-1 VNT. The expression vector is designed so that a VNT can be changed by routine cloning methods to produce a fusion polypeptide comprising IP-10 or MCP-3 fused with a Muc-1 VNT dimer, trimer, tetramer, pentamer, hexamer, etc.
In specific emobodiments, the present invention also provides a fusion polypeptide comprising human monocyte chemotactic protein-3 and human Muc-1, a fusion polypeptide comprising human interferon-induced protein 10 and human Muc-1, a fusion polypeptide comprising human macrophage-derived chemokine and human Muc-1, a fusion polypeptide comprising human SDF-1 and human Muc-1, a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:2, a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:1, a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:49 (human MDC fused to human Muc-1) and a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:54 (human SDF1 fused to human Muc-1).
The present invention further provides a fusion polypeptide comprising a human chemokine (e.g., IP-10, MCP-3, SDF-1, etc.) and a scFv which recognizes tumor antigens, such as idiotype-specific scFv, Muc-1, etc. Such a fusion polypeptide would allow migration, recruitment and activation of specialized cells of the immune system, such as natural killer (NK) cells, macrophages, dendritic cells (DC), polymorphonuclear (PMN) leukocytes, cytotoxic lymphocytes (CTL), etc., which would destroy the target cell.
The fusion polypeptide of this invention can further comprise a spacer sequence between the chemokine and the tumor antigen or viral antigen, which can have the amino acid sequence EFNDAQAPKSLE (SEQ ID NO:11), which allows for retention of the correct folding of the tumor antigen region of the polypeptide.
In addition, the present invention provides a composition comprising the fusion polypeptide of this invention and a suitable adjuvant. Such a composition can be in a pharmaceutically acceptable carrier, as described herein. As used herein, xe2x80x9csuitable adjuvantxe2x80x9d describes a substance capable of being combined with the fusion polypeptide to enhance an immune response in a subject without deleterious effect on the subject. A suitable adjuvant can be, but is not limited to, for example, an immunostimulatory cytokine, SYNTEX adjuvant formulation 1 (SAF-1) composed of 5 percent (wt/vol) squalene (DASF, Parsippany, N.J.), 2.5 percent Pluronic, L121 polymer (Aldrich Chemical, Milwaukee), and 0.2 percent polysorbate (Tween 80, Sigma) in phosphate-buffered saline. Other suitable adjuvants are well known in the art and include QS-21, Freund""s adjuvant (complete and incomplete), alum, aluminum phosphate, aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1xe2x80x2-2xe2x80x2-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE) and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trealose dimycolate and cell wall skeleton (MPL+TDM+CWS) in 2% squalene/Tween 80 emulsion. The adjuvant, such as an immunostimulatory cytokine can be administered before the administration of the fusion protein or nucleic acid encoding the fusion protein, concurrent with the administration of the fusion protein or nucleic acid or up to five days after the administration of the fusion polypeptide or nucleic acid to a subject. QS-21, similarly to alum, complete Freund""s adjuvant, SAF, etc., can be administered within hours of administration of the fusion protein.
Furthermore, combinations of adjuvants, such as immunostimulatory cytokines can be co-administered to the subject before, after or concurrent with the administration of the fusion polypeptide or nucleic acid. For example, combinations of adjuvants, such as immunostimulatory cytokines, can consist of two or more of immunostimulatory cytokines of this invention, such as GM/CSF, interleukin-2, interleukin-12, interferon-gamma, interleukin-4, tumor necrosis factor-alpha, interleukin-1, hematopoietic factor flt3L, CD40L, B7.1 co-stimulatory molecules and B7.2 co-stimulatory molecules. The effectiveness of an adjuvant or combination of adjuvants may be determined by measuring the immune response directed against the fusion polypeptide with and without the adjuvant or combination of adjuvants, using standard procedures, as described herein.
Furthermore, the present invention provides a composition comprising the fusion polypeptide of this invention or a nucleic acid encoding the fusion polypeptide of this invention and an adjuvant, such as an immunostimulatory cytokine or a nucleic acid encoding an adjuvant, such as an immunostimulatory cytokine. Such a composition can be in a pharmaceutically acceptable carrier, as described herein. The immunostimulatory cytokine used in this invention can be, but is not limited to, GM/CSF, interleukin-2, interleukin-12, interferon-gamma, interleukin-4, tumor necrosis factor-alpha, interleukin-1, hematopoietic factor flt3L, CD40L, B7.1 con-stimulatory molecules and B7.2 co-stimulatory molecules.
The present invention further contemplates a fusion polypeptide comprising a chemokine, or active fragment thereof, as described herein and an antigen of human immunodeficiency virus (HIV). For example, the HIV antigen of this invention can be, but is not limited to, the envelope glycoprotein gp120, the third hypervariable region of the envelope glycoprotein, gp120 of HIV-1 (the disulfate loop V3), having the amino acid sequence: NCTRPNNNTRKRIRIQRGPGRAFVTIGKIGNMRQAHCNIS (SEQ ID NO:10), any other antigenic fragment of gp120, the envelope glycoprotein gp160, an antigenic fragment of gp160, the envelope glycoprotein gp41 and an antigenic fragment of gp41. For example, the nucleic acid encoding the V3 loop can be fused to the 3xe2x80x2 end of the nucleic acid encoding a chemokine (e.g., IP-10, MCP-3, SDF-1, MDC) directly or separated by a spacer sequence. The chemokine-V3 loop fusion polypeptide can be produced in an expression system as described herein and purified as also described herein.
In specific embodiments, the present invention provides a fusion polypeptide comprising a human chemokine and a human immunodeficiency virus (HIV) antigen, wherein the chemokine can be IP-10, MCP-1, MCP-2, MCP-3, MCP-4, MIP 1, RANTES, SDF-1, MIG and/or MDC and wherein the HIV antigen can be gp120, gp160, gp41, an active (i.e., antigenic) fragment of gp120, an active (i.e., antigenic) fragment of gp160 and an active (i.e., antigenic) fragment of gp41.
Further provided in this invention is fusion polypeptide comprising human IP-10 and HIV gp120, a fusion polypeptide comprising human MCP-3 and HIV gp120, a fusion polypeptide comprising human MDC and HIV gp120, a fusion polypeptide comprising human SDF-1 and HIV gp120, a fusion polypeptide comprising the amino acid sequence of SEQ ED NO:6 (human IP-10/gp120), a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:7 (human MCP-3/gp120), a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:5 (human SDF1/gp120), a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:52, a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:56 and a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:50 (human MDC/gp120).
An isolated nucleic acid encoding the fusion polypeptides of this invention as described above is also provided. By xe2x80x9cisolated nucleic acidxe2x80x9d is meant a nucleic acid molecule that is substantially free of the other nucleic acids and other components commonly found in association with nucleic acid in a cellular environment. Separation techniques for isolating nucleic acids from cells are well known in the art and include phenol extraction followed by ethanol precipitation and rapid solubilization of cells by organic solvent or detergents (81).
The nucleic acid encoding the fusion polypeptide can be any nucleic acid that functionally encodes the fusion polypeptide. To functionally encode the polypeptide (i.e., allow the nucleic acid to be expressed), the nucleic acid can include, for example, expression control sequences, such as an origin of replication, a promoter, an enhancer and necessary information processing sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites and transcriptional terminator sequences. Preferred expression control sequences are promoters derived from metallothionine genes, actin genes, immunoglobulin genes, CMV, SV40, adenovirus, bovine papilloma virus, etc. A nucleic acid encoding a selected fusion polypeptide can readily be determined based upon the genetic code for the amino acid sequence of the selected fusion polypeptide and many nucleic acids will encode any selected fusion polypeptide. Modifications in the nucleic acid sequence encoding the fusion polypeptide are also contemplated. Modifications that can be useful are modifications to the sequences controlling expression of the fusion polypeptide to make production of the fusion polypeptide inducible or repressible as controlled by the appropriate inducer or repressor. Such means are standard in the art (81). The nucleic acids can be generated by means standard in the art, such as by recombinant nucleic acid techniques, as exemplified in the examples herein and by synthetic nucleic acid synthesis or in vitro enzymatic synthesis.
A vector comprising any of the nucleic acids of the present invention and a cell comprising any of the vectors of the present invention are also provided. The vectors of the invention can be in a host (e.g., cell line or transgenic animal) that can express the fusion polypeptide contemplated by the present invention.
There are numerous E. coli (Escherichia coli) expression vectors known to one of ordinary skill in the art useful for the expression of nucleic acid encoding proteins such as fusion proteins. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis, and other enterobacteria, such as Salmonella, Serratia, as well as various Pseudomonas species. These prokaryotic hosts can support expression vectors which will typically contain expression control sequences compatible with the host cell (e.g., an origin of replication). In addition, any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (Trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda. The promoters will typically control expression, optionally with an operator sequence and have ribosome binding site sequences for example, for initiating and completing transcription and translation. If necessary, an amino terminal methionine can be provided by insertion of a Met codon 5xe2x80x2 and in-frame with the protein. Also, the carboxy-terminal extension of the protein can be removed using standard oligonucleotide mutagenesis procedures.
Additionally, yeast expression can be used. There are several advantages to yeast expression systems. First, evidence exists that proteins produced in a yeast secretion system exhibit correct disulfide pairing. Second, post-translational glycosylation is efficiently carried out by yeast secretory systems. The Saccharomyces cerevisiae pre-pro-alpha-factor leader region (encoded by the MF xcex1-1 gene) is routinely used to direct protein secretion from yeast (82). The leader region of pre-pro-alpha-factor contains a signal peptide and a pro-segment which includes a recognition sequence for a yeast protease encoded by the KEX2 gene. This enzyme cleaves the precursor protein on the carboxyl side of a Lys-Arg dipeptide cleavage-signal sequence. The polypeptide coding sequence can be fused in-frame to the pre-pro-alpha-factor leader region. This construct is then put under the control of a strong transcription promoter, such as the alcohol dehydrogenase I promoter or a glycolytic promoter. The protein coding sequence is followed by a translation termination codon which is followed by transcription termination signals. Alternatively, the polypeptide coding sequence of interest can be fused to a second protein coding sequence, such as Sj26 or xcex2-galactosidase, used to facilitate purification of the fusion protein by affinity chromatography. The insertion of protease cleavage sites to separate the components of the fusion protein is applicable to constructs used for expression in yeast.
Efficient post-translational glycosylation and expression of recombinant proteins can also be achieved in Baculovirus systems in insect cells.
Mammalian cells permit the expression of proteins in an environment that favors important post-translational modifications such as folding and cysteine pairing, addition of complex carbohydrate structures and secretion of active protein. Vectors useful for the expression of proteins in mammalian cells are characterized by insertion of the protein coding sequence between a strong viral promoter and a polyadenylation signal. The vectors can contain genes conferring either gentamicin or methotrexate resistance for use as selectable markers. The antigen and immunoreactive fragment coding sequence can be introduced into a Chinese hamster ovary (CHO) cell line using a methotrexate resistance-encoding vector. Presence of the vector RNA in transformed cells can be confirmed by Northern blot analysis and production of a cDNA or opposite strand RNA corresponding to the protein coding sequence can be confirmed by Southern and Northern blot analysis, respectively. A number of other suitable host cell lines capable of secreting intact proteins have been developed in the art and include the CHO cell lines, HeLa cells, myeloma cell lines, Jurkat cells and the like. Expression vectors for these cells can include expression control sequences, as described above.
The vectors containing the nucleic acid sequences of interest can be transferred into the host cell by well-known methods, which vary depending on the type of cell host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment, lipofection or electroporation may be used for other cell hosts.
Alternative vectors for the expression of protein in mammalian cells, similar to those developed for the expression of human gamma-interferon, tissue plasminogen activator, clotting Factor VIII, hepatitis B virus surface antigen, protease Nexinl, and eosinophil major basic protein, can be employed. Further, the vector can include CMV promoter sequences and a polyadenylation signal available for expression of inserted nucleic acid in mammalian cells (such as COS7).
The nucleic acid sequences can be expressed in hosts after the sequences have been positioned to ensure the functioning of an expression control sequence. These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commnonly, expression vectors can contain selection markers, e.g., tetracycline resistance or hygromycin resistance, to permit detection and/or selection of those cells transformed with the desired nucleic acid sequences (83).
Additionally, the fusion polypeptides and/or nucleic acids of the present invention can be used in in vitro diagnostic assays, as well as in screening assays for identifying unknown tumor antigen epitopes and fine mapping of tumor antigen epitopes.
Also provided is a method for producing a fusion polypeptide comprising a chemokine, or an active fragment thereof and a tumor antigen or HIV antigen, comprising cloning into an expression vector a first DNA fragment encoding a chemokine or active fragment thereof and a second DNA fragment encoding a tumor antigen or HIV antigen; and expressing the DNA of the expression vector in an expression system under conditions whereby the fusion polypeptide is produced. The expression vector and expression system can be of any of the types as described herein. The cloning of the first and second DNA segments into the expression vector and expression of the DNA under conditions which allow for the production of the fusion protein of this invention can be carried out as described in the Examples section included herein. The method of this invention can further comprise the step of isolating and purifying the fusion polypeptide, according to methods well known in the art and as described herein.
Any of the fusion polypeptides, the nucleic acids and the vectors of the present invention can be in a pharmaceutically acceptable carrier and in addition, can include other medicinal agents, pharmaceutical agents, carriers, diluents, adjuvants (e.g., immunostimulatory cytokines), etc. By xe2x80x9cpharmaceutically acceptablexe2x80x9d is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the selected antigen without causing substantial deleterious biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art (84).
Thus, the present invention provides a method for inducing an immune response in a subject capable of induction of an immune response and preferably human, comprising administering to the subject an immune response-inducing amount of the fusion polypeptide of this invention. As used herein, xe2x80x9can immune response-inducing amountxe2x80x9d is that amount of fusion polypeptide which is capable of producing in a subject a humoral and/or cellular immune response capable of being detected by standard methods of measurement, such as, for example, as described herein. For example, the antigenic polypeptide region can induce an antibody response. The antibodies can treat or prevent a pathological or harmful condition in the subject in which the antibodies are produced or the antibodies can be removed from the subject and administered to another subject to treat or prevent a pathological or harmful condition. The fusion polypeptide can also induce an effector T cell (cellular) immune response which is effective in treating or preventing a pathological or harmful conditions in the subject.
In an embodiment wherein the antigen moiety of the fusion polypeptide comprises an immunoglobulin light or heavy chain or a single chain antibody, the immune response can be the production in the subject of anti-idiotype antibodies, which represent the image of the original antigen and can function in a vaccine preparation to induce an immune response to a pathogenic antigen, thereby avoiding immunization with the antigen itself (85). The anti-idiotype antibodies can treat or prevent a pathological or harmful condition in the subject in which the anti-idiotype antibodies are produced or the anti-idiotype antibodies can be removed from the subject and administered to another subject to treat or prevent a pathological or harmful condition.
Further provided is a method for inhibiting the growth of tumor cells in a subject, comprising administering to the subject a tumor cell growth-inhibiting amount of the fusion polypeptide of this invention. The subject of this method can be any subject in which a humoral and/or cellular immune response to a tumor can be induced, which is preferably an animal and most preferably a human. As used herein, xe2x80x9cinhibiting the growth of tumor cellsxe2x80x9d means that following administration of the fusion polypeptide, a measurable humoral and/or cellular immune response against the tumor cell epitope is elicited in the subject, resulting in the inhibition of growth of tumor cells present in the subject. The humoral immune response can be measured by detection, in the serum of the subject, of antibodies reactive with the epitope of the tumor antigen present on the fusion polypeptide, according to protocols standard in the art, such as enzyme linked immunosorbent immunoassay (ELISA) and Western blotting protocols. The cellular immune response can be measured by, for example, footpad swelling in laboratory animals, peripheral blood lymphocyte (PBL) proliferation assays and PBL cytotoxicity assays, as would be known to one of ordinary skill in the art of immunology and particularly as set forth in the available handbooks and texts of immunology protocols (86).
The present invention also provides a method of treating cancer in a subject diagnosed with cancer, comprising administering to the subject an effective amount of the fusion polypeptide of the present invention. The cancer can be, but is not limited to B cell lymphoma, T cell lymphoma, myeloma, leukemia, breast cancer, pancreatic cancer, colon cancer, lung cancer, renal cancer, liver cancer, prostate cancer, melanoma and cervical cancer.
Further provided is a method of treating a B cell tumor in a subject diagnosed with a B cell tumor, comprising administering an effective amount of the fusion polypeptide of this invention, which comprises an antibody or a fragment thereof, as described herein, in a pharmaceutically acceptable carrier, to the subject.
In specific embodiments, the present invention also provides a method of producing an immune response in a subject, comprising administering to the subject a composition comprising a fusion polypeptide of this invention and a pharmaceutically acceptable carrier and wherein the fusion polypeptide can be a fusion polypeptide comprising human monocyte chemotactic protein-3 and human Muc-1, a fusion polypeptide comprising human interferon-induced protein 10 and human Muc-1, a fusion polypeptide comprising human macrophage-derived chemokine and human Muc-1, a fusion polypeptide comprising human SDF-1 and human Muc-1, a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:2, a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:1, a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:49 and a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:54, thereby producing an immune response in the subject.
Also provided is a method of producing an immune response in a subject, comprising administering to the subject a composition comprising a nucleic acid encoding a fusion polypeptide of this invention and a pharmaceutically acceptable carrier and wherein the fusion polypeptide is a fusion polypeptide comprising comprising human monocyte chemotactic protein-3 and human Muc-1, a fusion polypeptide comprising human interferon-induced protein 10 and human Muc-1, a fusion polypeptide comprising human macrophage-derived chemokine and human Muc-1, a fusion polypeptide comprising human SDF-1 and human Muc-1, a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:2, a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:1, a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:49 and a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:54, under conditions whereby the nucleic acid of the composition can be expressed, thereby producing an immune response in the subject.
In further embodiments, the present invention also provides a method of producing an immune response in a subject, comprising administering to the subject a composition comprising a fusion polypeptide of this invention and a pharmaceutically acceptable carrier and wherein the fusion polypeptide can be a fusion polypeptide comprising human IP-10 and HIV gp120, a fusion polypeptide comprising human MCP-3 and HIV gp120, a fusion polypeptide comprising human MDC and HIV gp120, a fusion polypeptide comprising human SDF-1 and HIV gp120, a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:6, a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:7, a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:52, a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:56, a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:5, and/or a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:50, thereby producing an immune response in the subject.
Also provided is a method of producing an immune response in a subject, comprising administering to the subject a composition comprising a nucleic acid encoding a fusion polypeptide of this invention and a pharmaceutically acceptable carrier and wherein the fusion polypeptide is a fusion polypeptide comprising human IP-10 and HIV gp120, a fusion polypeptide comprising human MCP-3 and HIV gp120, a fusion polypeptide comprising human MDC and HIV gp120, a fusion polypeptide comprising human SDF-1 and HIV gp120, a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:6, a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:7, a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:5, a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:52, a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:56, and/or a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:50, under conditions whereby the nucleic acid of the composition can be expressed, thereby producing an immune response in the subject.
Also provided is a method of producing an immune response in a subject, comprising administering to the subject a composition comprising a fusion polypeptide and a pharmaceutically acceptable carrier and wherein the fusion polypeptide is a fusion polypeptide comprising a human chemokine and a human immunodeficiency virus (HIV) antigen, wherein the chemokine can be IP-10, MCP-1, MCP-2, MCP-3, MCP-4, MIP 1, RANTES, SDF-1, MIG and/or MDC and wherein the HIV antigen can be gp120, gp160, gp41, an active (i.e., antigenic) fragment of gp120, an active (i.e., antigenic) fragment of gp160 and/or an active (i.e., antigenic) fragment of gp41, thereby producing an immune response in the subject.
The present invention also provides a method of producing an immune response in a subject, comprising administering to the subject a composition comprising a nucleic acid encoding a fusion polypeptide comprising a human chemokine and a human immunodeficiency virus (HIV) antigen, wherein the chemokine can be IP-10, MCP-1, MCP-2, MCP-3, MCP-4, MIP 1, RANTES, SDF-1, MIG and/or MDC and wherein the HIV antigen can be gp120, gp160, gp41, an active (i.e., antigenic) fragment of gp120, an active (i.e., antigenic) fragment of gp160 and/or an active (i.e., antigenic) fragment of gp41, and a pharmaceutically acceptable carrier, under conditions whereby the nucleic acid can be expressed, thereby producing an immune response in the subject.
In any of the methods provided herein which recite the production of an immune response, the immune response can be humoral and/or an effector T cell (cellular) immune response, as determined according to methods standard in the art.
In another embodiment, the present invention provides a method of treating a cancer in a subject comprising adminstering to the subject a composition comprising a fusion polypeptide of this invention and a pharmaceutically acceptable carrier and wherein the fusion polypeptide is a fusion polypeptide comprising human monocyte chemotactic protein-3 and human Muc-1, a fusion polypeptide comprising human interferon-induced protein 10 and human Muc-1, a fusion polypeptide comprising human macrophage-derived chemokine and human Muc-1, a fusion polypeptide comprising human SDF-1 and human Muc-1, a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:2, a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:1, a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:49 and a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:54, thereby treating a cancer in the subject.
Additionally provided is a method of treating a cancer in a subject, comprising administering to the subject a composition comprising a nucleic acid encoding a fusion polypeptide of this invention and a pharmaceutically acceptable carrier and wherein the fusion polypeptide is a fusion polypeptide comprising human monocyte chemotactic protein-3 and human Muc-1, a fusion polypeptide comprising human interferon-induced protein 10 and human Muc-1, a fusion polypeptide comprising human macrophage-derived chemokine and human Muc-1, a fusion polypeptide comprising human SDF-1 and human Muc-1, a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:2, a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:1, a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:49 and a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:54, under conditions whereby the nucleic acid of the composition can be expressed, thereby treating a cancer in the subject.
Further provided is a method of treating or preventing HIV infection in a subject, comprising administering to the subject a composition comprising a human chemokine and a human immunodeficiency virus (HIV) antigen, wherein the chemokine can be IP-10, MCP-1, MCP-2, MCP-3, MCP-4, MIP 1, RANTES, SDF-1, MIG and/or MDC and wherein the HIV antigen can be gp120, gp160, gp41, an active (i.e., antigenic) fragment of gp120, an active (i.e., antigenic) fragment of gp160 and/or an active (i.e., antigenic) fragment of gp41, and a pharmaceutically acceptable carrier, thereby treating or preventing HIV infection in the subject.
In addition, a method of treating or preventing HIV infection in a subject is provided herein, comprising administering to the subject a composition comprising a nucleic acid encoding a fusion polypeptide comprising a human chemokine and a human immunodeficiency virus (HIV) antigen, wherein the chemokine can be IP-10, MCP-1, MCP-2, MCP-3, MCP-4, MIP 1, RANTES, SDF-1, MIG and/or MDC and wherein the HIV antigen can be gp120, gp160, gp41, an active (i.e., antigenic) fragment of gp120, an active (i.e., antigenic) fragment of gp160 and/or an active (i.e., antigenic) fragment of gp41, and a pharmaceutically acceptable carrier, under conditions whereby the nucleic acid can be expressed, thereby treating or preventing HIV infection in the subject.
Further provided is a method of treating or preventing HIV infection in a subject, comprising administering to the subject a composition comprising a fusion polypeptide comprising human IP-10 and HIV gp120, a fusion polypeptide comprising human MCP-3 and HIV gp120, a fusion polypeptide comprising human MDC and HIV gp120, a fusion polypeptide comprising human SDF-1 and HIV gp120, a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:6, a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:7, a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:5, a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:52, a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:56 and/or a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:50, and a pharmaceutically acceptable carrier, thereby treating or preventing HIV infection in the subject.
In addition, a method of treating or preventing HIV infection in a subject is provided herein, comprising administering to the subject a composition comprising a nucleic acid encoding a fusion polypeptide comprising human IP-10 and HIV gp120, a fusion polypeptide comprising human MCP-3 and HIV gp120, a fusion polypeptide comprising human MDC and HIV gp120, a fusion polypeptide comprising human SDF-1 and HIV gp120, a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:6, a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:7, a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:5, a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:52, a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:56, and/or a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:50, and a pharmaceutically acceptable carrier, under conditions whereby the nucleic acid can be expressed, thereby treating or preventing HIV infection in the subject.
In a further embodiment, the present invention provides a method of treating a B cell tumor in a subject, comprising administering to the subject a fusion polypeptide comprising a human chemokine and a B cell tumor antigen, wherein the B cell tumor antigen can be an antibody, a single chain antibody or an epitope of an idiotype of an antibody, wherein the human chemokine can be MCP-3, MDC or SDF-1, wherein the fusion polypeptide can be a fusion polypeptide comprising human MCP-3 and human a single chain antibody, a fusion polypeptide comprising human MDC and a human single chain antibody or a fusion polypeptide comprising human SDF-1 and a human single chain antibody and wherein the fusion polypeptide can be a polypeptide having the amino acid sequence of SEQ ID NO:51 (human MCP-3/human scFV fusion), a polypeptide having the amino acid sequence of SEQ ID NO:53 (human MDC/human scFv fusion) and/or a polypeptide having the amino acid sequence of SEQ ID NO:55 (human SDF-1/human scFv fusion), thereby treating a B cell tumor in the subject.
Also provided is a fusion polypeptide comprising the human chemokine, SDF-1xcex2, and the V3 loop of HIV-1 envelope glycoprotein, gp120, as well as a fusion protein comprising SDF-1 and gp160 of HIV-1, a fusion protein comprising SDF-1xcex2 and gp41 of HIV-1, a fusion protein comprising SDF-1xcex2 and an active fragment of gp120, a fusion protein comprising SDF-1xcex2 and an active fragment of gp160 and a fusion polypeptide comprising SDF-1xcex2 and an active fragment of gp41.
The methods of this invention comprising administering the fusion protein of this invention to a subject can further comprise the step of administering one or more adjuvants, such as an immunostimulatory cytokine to the subject. The adjuvant or adjuvants can be administered to the subject prior to, concurrent with and/or after the administration of the fusion protein as described herein.
The subject of the present invention can be any animal in which cancer can be treated by eliciting an immune response to a tumor antigen. In a preferred embodiment, the animal is a mammal and most preferably is a human.
To determine the effect of the administration of the fusion polypeptide on inhibition of tumor cell growth in laboratory animals, the animals can either be pre-treated with the fusion polypeptide and then challenged with a lethal dose of tumor cells, or the lethal dose of tumor cells can be administered to the animal prior to receipt of the fusion polypeptide and survival times documented. To determine the effect of administration of the fusion polypeptide on inhibition of tumor cell growth in humans, standard clinical response parameters can be analyzed.
To determine the amount of fusion polypeptide which would be an effective tumor cell growth-inhibiting amount, animals can be treated with tumor cells as described herein and varying amounts of the fusion polypeptide can be administered to the animals. Standard clinical parameters, as described herein, can be measured and that amount of fusion polypeptide effective in inhibiting tumor cell growth can be determined. These parameters, as would be known to one of ordinary skill in the art of oncology and tumor biology, can include, but are not limited to, physical examination of the subject, measurements of tumor size, X-ray studies and biopsies.
The present invention further provides a method for treating or preventing HIV infection in a human subject, comprising administering to the subject an HIV replication-inhibiting amount of the chemokine/HIV antigen fusion polypeptide of this invention. As used herein, xe2x80x9ca replication-inhibiting amountxe2x80x9d is that amount of fusion polypeptide which produces a measurable humoral and/or effector T cell (cellular) immune response in the subject against the viral antigen, as determined by standard immunological protocols, resulting in the inhibition of HIV replication in cells of the subject, as determined by methods well known in the art for measuring HIV replication, such as viral load measurement, which can be determined by quantitative PCR (QPCR) and branched DNA (bDNA) analysis; reverse transcriptase activity measurement, in situ hybridization, Western immunoblot, ELISA and p24 gag measurement (87,88,89,90,91). The fusion polypeptide can be administered to the subject in varying amounts and the amount of the fusion polypeptide optimally effective in inhibiting HIV replication in a given subject can be determined as described herein.
The fusion polypeptide of this invention can be administered to the subject orally or parenterally, as for example, by intramuscular injection, by intraperitoneal injection, topically, transdermally, injection directly into the tumor, or the like, although subcutaneous injection is typically preferred. Immunogenic, tumor cell growth inhibiting and HIV replication inhibiting amounts of the fusion polypeptide can be determined using standard procedures, as described. Briefly, various doses of the fusion polypeptide are prepared, administered to a subject and the immunological response to each dose is determined (92). The exact dosage of the fusion polypeptide will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the cancer or HIV infection that is being treated, the particular antigen being used, the mode of administration, and the like. Thus, it is not possible to specify an exact amount. However, an appropriate amount may be determined by one of ordinary skill in the art using only routine screening given the teachings herein.
Generally, the dosage of fusion protein will approximate that which is typical for the administration of vaccines, and typically, the dosage will be in the range of about 1 to 500 xcexcg of the fusion polypeptide per dose, and preferably in the range of 50 to 250 xcexcg of the fusion polypeptide per dose. This amount can be administered to the subject once every other week for about eight weeks or once every other month for about six months. The effects of the administration of the fusion polypeptide can be determined starting within the first month following the initial administration and continued thereafter at regular intervals, as needed, for an indefinite period of time.
For oral administration of the fusion polypeptide of this invention, fine powders or granules may contain diluting, dispersing, and/or surface active agents, and may be presented in water or in a syrup, in capsules or sachets in the dry state, or in a nonaqueous solution or suspension wherein suspending agents may be included, in tablets wherein binders and lubricants may be included, or in a suspension in water or a syrup. Where desirable or necessary, flavoring, preserving, suspending, thickening, or emulsifying agents may be included. Tablets and granules are preferred oral administration forms, and these may be coated.
Parenteral administration, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system, such that a constant level of dosage is maintained. See, e.g., U.S. Pat. No. 3,710,795, which is incorporated by reference herein.
For solid compositions, conventional nontoxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like. Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, etc. an active compound as described herein and optional pharmaceutical adjuvants in an excipient, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form a solution or suspension. If desired, the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, etc. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this (84).
The present invention also provides a method for producing single chain antibodies against tumor antigens comprising producing a fusion polypeptide comprising a chemokine region and a region comprising a tumor antigen; immunizing animals with an amount of the fusion polypeptide sufficient to produce a humoral immune response to the fusion polypeptide; isolating spleen cells expressing immunoglobulin specific for the fusion polypeptide; isolating the immunoglobulin variable genes from the spleen cells; cloning the immunoglobulin variable genes into an expression vector; expressing the immunoglobulin variable genes in a bacteriophage; infecting E. coli cells with the bacteriophage; isolating bacteriophage from the E. coli cells which express the immunoglobulin variable genes and isolating the immunoglobulin variable gene products for use as single chain antibodies.
The chemokine-scFv fusion proteins described herein would be better targets than tumor cells or purified tumor antigen peptides for antibody selection approaches such as phage displayed scFv production. For example, there are two ways to produce specific Fv displayed on the surface of phage: (1) Immunize mice with tumor cells; isolate immunoglobulin variable fragment genes from spleen cells by RT/PCR; clone the genes into bacteriophage in frame with genes coding phage surface proteins (e.g., major coat protein subunits gpVIII or gpIII of the filamentous bacteriophage) (93,94); and (2) Construct semisynthetic antibody libraries by PCR as described (95). The specific phage producing scFv are selected by several rounds of binding elution and infection in E. coli, using biotin labeled chemokine-tumor antigen (e.g., Muccore). The biotin enables selection of high affinity scFv-phage through binding to streptavidin conjugated magnetic beads. This approach provides simple, fast and efficient production of specific anti-tumor epitope scFv.
As described herein, the present invention also provides a nucleic acid which encodes a fusion polypeptide of this invention and a vector comprising a nucleic acid which encodes a fusion polypeptide of this invention, either of which can be in a pharmaceutically acceptable carrier. Such nucleic acids and vectors can be used in gene therapy protocols to treat cancer as well as to treat or prevent HIV infection in a subject.
Thus, the present invention further provides a method of treating a cancer in a subject diagnosed with a cancer comprising administering the nucleic acid of this invention to a cell of the subject under conditions whereby the nucleic acid is expressed in the cell, thereby treating the cancer.
A method of treating a B cell tumor in a subject diagnosed with a B cell tumor is also provided, comprising administering the nucleic acid of this invention, encoding a chemokine and an antibody or fragment thereof, in a pharmaceutically acceptable carrier, to a cell of the subject, under conditions whereby the nucleic acid is expressed in the cell, thereby treating the B cell tumor.
The methods of this invention comprising administering nucleic acid encoding the fusion protein of this invention to a subject can further comprise the step of administering a nucleic acid encoding an adjuvant such as an immunostimulatory cytokine to the subject, either before, concurrent with or after the administration of the nucleic acid encoding the fusion protein, as described herein.
The nucleic acid can be administered to the cell in a virus, which can be, for example, adenovirus, retrovirus and adeno-associated virus. Alternatively, the nucleic acid of this invention can be administered to the cell in a liposome. The cell of the subject can be either in vivo or ex vivo. Also, the cell of the subject can be any cell which can take up and express exogenous nucleic acid and produce the fusion polypeptide of this invention. Thus, the fusion polypeptide of this invention can be produced by a cell which secretes it, whereby it binds a chemokine receptor and is subsequently processed by an antigen presenting cell and presented to the immune system for elicitation of an immune response. Alternatively, the fusion polypeptide of this invention can be produced in an antigen presenting cell where it is processed directly and presented to the immune system.
If ex vivo methods are employed, cells or tissues can be removed and maintained outside the body according to standard protocols well known in the art. The nucleic acids of this invention can be introduced into the cells via any gene transfer mechanism, such as, for example, virus-mediated gene delivery, calcium phosphate mediated gene delivery, electroporation, microinjection or proteoliposomes. The transduced cells can then be infused (e.g., in a pharmaceutically acceptable carrier) or transplanted back into the subject per standard methods for the cell or tissue type. Standard methods are known for transplantation or infusion of various cells into a subject.
For in vivo methods, the nucleic acid encoding the fusion protein can be administered to the subject in a pharmaceutically acceptable carrier as described herein.
In the methods described herein which include the administration and uptake of exogenous DNA into the cells of a subject (i.e., gene transduction or transfection), the nucleic acids of the present invention can be in the form of naked DNA or the nucleic acids can be in a vector for delivering the nucleic acids to the cells for expression of the nucleic acid to produce the fusion protein of this invention. The vector can be a commercially available preparation, such as an adenovirus vector (Quantum Biotechnologies, Inc. (Laval, Quebec, Canada). Delivery of the nucleic acid or vector to cells can be via a variety of mechanisms. As one example, delivery can be via a liposome, using commercially available liposome preparations such as LIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, Md.), SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison, Wis.), as well as other liposomes developed according to procedures standard in the art. In addition, the nucleic acid or vector of this invention can be delivered in vivo by electroporation, the technology for which is available from Genetronics, Inc. (San Diego, Calif.) as well as by means of a SONOPORATION machine (ImaRx Pharmaceutical Corp., Tucson, Ariz.).
Vector delivery can also be via a viral system, such as a retroviral vector system which can package a recombinant retroviral genome (see e.g., 96,97). The recombinant retrovirus can then be used to infect and thereby deliver to the infected cells nucleic acid encoding the fusion polypeptide. The exact method of introducing the exogenous nucleic acid into mammalian cells is, of course, not limited to the use of retroviral vectors. Other techniques are widely available for this procedure including the use of adenoviral vectors (98), adeno-associated viral (AAV) vectors (99), lentiviral vectors (100), pseudotyped retroviral vectors (101). Physical transduction techniques can also be used, such as liposome delivery and receptor-mediated and other endocytosis mechanisms (see, for example, 102). This invention can be used in conjunction with any of these or other commonly used gene transfer methods.
Various adenoviruses may be used in the compositions and methods described herein. For example, a nucleic acid encoding the fusion protein can be inserted within the genome of adenovirus type 5. Similarly, other types of adenovirus may be used such as type 1, type 2, etc. For an exemplary list of the adenoviruses known to be able to infect human cells and which therefore can be used in the present invention, see Fields, et al. (103). Furthermore, it is contemplated that a recombinant nucleic acid comprising an adenoviral nucleic acid from one type adenovirus can be packaged using capsid proteins from a different type adenovirus.
The adenovirus of the present invention is preferably rendered replication deficient, depending upon the specific application of the compounds and methods described herein. Methods of rendering an adenovirus replication deficient are well known in the art. For example, mutations such as point mutations, deletions, insertions and combinations thereof, can be directed toward a specific adenoviral gene or genes, such as the E1 gene. For a specific example of the generation of a replication deficient adenovirus for use in gene therapy, see WO 94/28938 (Adenovirus Vectors for Gene Therapy Sponsorship) which is incorporated herein in its entirety.
In the present invention, the nucleic acid encoding the fusion protein can be inserted within an adenoviral genome and the fusion protein encoding sequence can be positioned such that an adenovirus promoter is operatively linked to the fusion protein nucleic acid insert such that the adenoviral promoter can then direct transcription of the nucleic acid, or the fusion protein insert may contain its own adenoviral promoter. Similarly, the fusion protein insert may be positioned such that the nucleic acid encoding the fusion protein may use other adenoviral regulatory regions or sites such as splice junctions and polyadenylation signals and/or sites. Alternatively, the nucleic acid encoding the fusion protein may contain a different enhancer/promoter (e.g., CMV or RSV-LTR enhancer/promoter sequences) or other regulatory sequences, such as splice sites and polyadenylation sequences, such that the nucleic acid encoding the fusion protein may contain those sequences necessary for expression of the fusion protein and not partially or totally require these regulatory regions and/or sites of the adenovirus genome. These regulatory sites may also be derived from another source, such as a virus other than adenovirus. For example, a polyadenylation signal from SV40 or BGH may be used rather than an adenovirus, a human, or a murine polyadenylation signal. The fusion protein nucleic acid insert may, alternatively, contain some sequences necessary for expression of the nucleic acid encoding the fusion protein and derive other sequences necessary for the expression of the fusion protein nucleic acid from the adenovirus genome, or even from the host in which the recombinant adenovirus is introduced.
As another example, for administration of nucleic acid encoding the fusion protein to an individual in an AAV vector, the AAV particle can be directly injected intravenously. The AAV has a broad host range, so the vector can be used to transduce any of several cell types, but preferably cells in those organs that are well perfused with blood vessels. To more specifically administer the vector, the AAV particle can be directly injected into a target organ, such as muscle, liver or kidney. Furthermore, the vector can be administered intraarterially, directly into a body cavity, such as intraperitoneally, or directly into the central nervous system (CNS).
An AAV vector can also be administered in gene therapy procedures in various other formulations in which the vector plasmid is administered after incorporation into other delivery systems such as liposomes or systems designed to target cells by receptor-mediated or other endocytosis procedures. The AAV vector can also be incorporated into an adenovirus, retrovirus or other virus which can be used as the delivery vehicle.
As described above, the nucleic acid or vector of the present invention can be administered in vivo in a pharmaceutically acceptable carrier. By xe2x80x9cpharmaceutically acceptablexe2x80x9d is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.
The mode of administration of the nucleic acid or vector of the present invention can vary predictably according to the disease being treated and the tissue being targeted. For example, for administration of the nucleic acid or vector in a liposome, catheterization of an artery upstream from the target organ is a preferred mode of delivery, because it avoids significant clearance of the liposome by the lung and liver.
The nucleic acid or vector may be administered orally as described herein for oral administration of the fusion polypeptides of this invention, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, topically or the like, although intravenous administration is typically preferred. The exact amount of the nucleic acid or vector required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every nucleic acid or vector. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein (84).
As one example, if the nucleic acid of this invention is delivered to the cells of a subject in an adenovirus vector, the dosage for administration of adenovirus to humans can range from about 107 to 109 plaque forming units (pfu) per injection, but can be as high as 1012 pfu per injection (104,105). Ideally, a subject will receive a single injection. If additional injections are necessary, they can be repeated at six month intervals for an indefinite period and/or until the efficacy of the treatment has been established.
Parenteral administration of the nucleic acid or vector of the present invention, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Pat. No. 3,610,795, which is incorporated by reference herein in its entirety.